PROJECT SUMMARY Drosophila research labs routinely maintain hundreds to thousands of unique strains, where each new generation must be transferred to fresh food every 4 to 6 weeks. Not only is this practice labor and resource intensive, but there is a risk that valuable strains will be lost or acquire additional mutations. This limitation would be overcome by the development of Drosophila cryopreservation methods. Thus, the overall goal of this proposal is to develop a simple, robust method to cryopreserve Drosophila embryos. Based on literature review and our expertise in cryobiology and Drosophila biology, we anticipate that vitrification is the optimal approach to cryopreservation. Vitrification is an `ice-free' method of cryopreservation where cells are loaded with high concentrations of cryoprotective agents (CPAs, e.g. dimethyl sulfoxide, propylene glycol, etc.) and rapidly cooled through the glass transition. The result is formation of an amorphous glassy state as opposed to crystalline ice. Nearly thirty years ago, a vitrification protocol for wild-type Drosophila was reported. However, this protocol is quite complex and results in a loss of nearly 70% of embryos upon development into adults. However, this breakthrough method represents an excellent starting point for us here, where we will take a comprehensive approach to both simplify and optimize the vitrification procedure by incorporating concepts and technologies from industrial chemistry, engineering and cryobiology. In Aim 1, we will simplify the embryo permeabilization procedure and then automate the liquid handling steps of the protocol by incorporating microfluidics. Automation will reduce user-error and decrease labor demands. In Aim 2, we will optimize the vitrification procedure by systematically evaluating a series of CPAs and CPA cocktail solutions. This information will then be used to increase the intraembryonic concentration of CPAs, thereby reducing the risk of ice crystallization upon cooling or rewarming. We will finally merge results from both Aims to validate this simplified and optimized vitrification procedure, where our goal is to achieve ?60% survival of thawed embryos to adults. To better ensure the successful completion the work described here, we have assembled a multidisciplinary team including cryobiologists, engineers and Drosophila biology experts. The successful completion of this exploratory proposal will result in the development of a simplified and robust vitrification method for Drosophila embryos that can be quickly implemented into laboratories and resource centers.